Archaeobacterial Ether Lipid Liposomes (Archaeosomes) as Novel Vaccine and Drug Delivery Systems

ABSTRACT:?

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeo-bacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.     

Investigation of parameters influencing incorporation,retention and cellular cytotoxicity in liposomal formulations of poorly soluble camptothecin

Abstract

Improving tumor delivery of lipophilic drugs through identifying advanced drug carrier systems with efficient carrier potency is of high importance. We have performed an investigative approach to identify parameters that affect liposomes’ ability to effectively deliver lipophilic camptothecin (CPT) to target cells. CPT is a potent anticancer drug, but its undesired physiological properties are impairing its therapeutic use. In this study, we have identified parameters influencing incorporation and retention of lipophilic CPT in liposomes, evaluating the effect of lipid composition, lipid chemical structure (head and tail group variations, polymer inclusion), zeta potential and anisotropy. Polyethyleneglycol (PEG) surface decoration was included to avoid liposome fusing and increase the potential for prolonged in vivo circulation time. The in vitro effect of the different carrier formulations on cell cytotoxicity was compared and the effect of active targeting of one of the formulations was evaluated. We found that a combination of liposome surface charge, lipid headgroup and carbon chain unsaturation affect CPT incorporation. Retention in liposomes was highly dependent on the liposomal surroundings and liposome zeta potential. Inclusion of lipid tethered PEG provided stability and prevented liposome fusing. PEGylation negatively affected CPT incorporation while improving retention. In vitro cell culture testing demonstrated that all formulations increased CPT potency compared to free CPT, while cationic formulations proved significantly more toxic to cancer cells that healthy cells. Finally, antibody mediated targeting of one liposome formulation further enhanced the selectivity towards targeted cancer cells, rendering normal cells fully viable after 1 hour exposure to targeted liposomes.     

Analysis and Optimization of the Cationic Lipid Component of a Lipid/Peptide Vector Formulation for Enhanced Transfection In Vitro and In Vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K₁₆GACRRETAWACG) and Lipofectin^®, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Novel Camptothecin Analogue (Gimatecan)‐Containing Liposomes Prepared by the Ethanol Injection Method

Small‐sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug‐containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1‐palmitoyl‐2‐oleoyl‐sn‐glycero‐3‐phosphocholine) then on the novel L‐carnitine derivative PUCE (palmitoyl‐(R)‐carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of “multiple injection method” was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan‐containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI‐H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no‐liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water‐insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.     

Impact of Apoplast Volume on Ionic Relations in Plant Cells

This study was performed to investigate the effect of cholesterol content, surface charge and sterical stabilization on the physico-chemical properties of liposomes prepared from the cancerostatic alkylphospholipid, octadecyl-1,1-dimethyl-piperidino-4-yl-phosphate (D21266), and their relationship to in vitro cytotoxicity. Stable incorporation of OPP into liposomes was found to be highly dependent on the cholesterol content. ³¹P-NMR spectroscopy as well as analysis of the lipid composition of OPP-containing liposome formulations revealed an increase in the amount of non-liposome-associated, micellar OPP as the cholesterol content decreased. The fraction of non-liposome-associated OPP constituted about 10% of total OPP when cholesterol was present in equimolar amounts (45.5/45.5 mol %) and increased to approximately 30% at a twofold excess of OPP over cholesterol (58.8/29.4 mol %). In monolayer incorporation studies it was shown that the existence of an increasing micellar pool of lipids leads to increased lipid transfer into the target monolayer. Liposome formulations containing more OPP than cholesterol were also found to display greater cytotoxicity. However, all liposome formulations were less cytotxic than pure (micellar) OPP. Cytotoxicity was not affected by the incorporation of N-methoxy-polyethyleneglycol₂₀₀₀-phosphoethanolamine, a lipid that is known to reduce liposome uptake into phagocytic cells. The results demonstrate that the increase in cell toxicity correlates with the increase in non-liposome-associated, micellar OPP, which can readily exchange into cellular membranes. Received: 4 October 2000/Revised: 29 March 2001     

Nanosized self-emulsifying lipid vesicles of diacylglycerol-PEG lipid conjugates: Biophysical characterization and inclusion of lipophilic dietary supplements

Hydrated diacylglycerol-PEG lipid conjugates, glyceryl dioleate-PEG12 (GDO-PEG12) and glyceryl dipalmitate-PEG23 (GDP-PEG23), spontaneously form uni- or oligolamellar liposomes in their liquid crystalline phase, in distinct difference from the PEGylated phospholipids which form micelles. GDP-PEG23 exhibits peculiar hysteretic phase behavior and can arrange into a long-living hexagonal phase at ambient and physiological temperatures. Liposomes of GDO-PEG12 and its mixture with soy lecithin exchange lipids with the membranes much more actively than common lecithin liposomes; such an active lipid exchange might facilitate the discharging of the liposome cargo upon uptake and internalization, and can thus be important in drug delivery applications. Diacylglycerol-PEG lipid liposome formulations can encapsulate up to 20-30 wt.% lipophilic dietary supplements such as fish oil, coenzyme Q10, and vitamins D and E. The encapsulation is feasible by way of dry mixing, avoiding the use of organic solvent.     

Fluorescent lipid probes: some properties and applications (a review)

Odd as it may seem, experimental challenges in lipid research are often hampered by the simplicity of the lipid structure. Since, as in protein research, mutants or overexpression of lipids are not realistic, a considerable amount of lipid research relies on the use of tagged lipid analogues. However, given the size of an average lipid molecule, special care is needed for the selection of probes, since if the size and intramolecular localization of the probe is not specifically taken into account, it may dramatically affect the properties of the lipids. The latter is particularly important in cell biological studies of lipid trafficking and sorting, where the probed lipid should resemble its natural counterpart as closely as possible. On the other hand, for biophysical applications, these considerations may be less critical. Here we provide a brief overview of the application of several lipid probes in cell biological and biophysical research, and critically analyze their validity in the various fields.     

Archaeobacterial ether lipid liposomes (archaeosomes) as novel vaccine and drug delivery systems

Liposomes are artificial, spherical, closed vesicles consisting of one or more lipid bilayer(s). Liposomes made from ester phospholipids have been studied extensively over the last 3 decades as artificial membrane models. Considerable interest has been generated for applications of liposomes in medicine, including their use as diagnostic reagents, as carrier vehicles in vaccine formulations, or as delivery systems for drugs, genes, or cancer imaging agents. The objective of this article is to review the properties and potential applications of novel liposomes made from the membrane lipids of Archaeobacteria (Archaea). These lipids are unique and distinct from those encountered in Eukarya and Bacteria. Polar glycerolipids make up the bulk of the membrane lipids, with the remaining neutral lipids being primarily squalenes and other hydrocarbons. The polar lipids consist of regularly branched, and usually fully saturated, phytanyl chains of 20, 25, or 40 carbon length, with the 20 and 40 being most common. The phytanyl chains are attached via ether bonds to the sn-2,3 carbons of the glycerol backbone(s). It has been shown only recently that total polar lipids of archaeobacteria, and purified lipid fractions therefrom, can form liposomes. We refer to liposomes made with any lipid composition that includes ether lipids characteristic of Archaeobacteria as archaeosomes to distinguish them from vesicles made from the conventional lipids obtained from eukaryotic or eubacterial sources or their synthetic analogs. In general, archaeosomes demonstrate relatively higher stabilities to oxidative stress, high temperature, alkaline pH, action of phospholipases, bile salts, and serum proteins. Some archaeosome formulations can be sterilized by autoclaving, without problems such as fusion or aggregation of the vesicles. The uptake of archaeosomes by phagocytic cells can be up to 50-fold greater than that of conventional liposome formulations. Studies in mice have indicated that systemic administration of several test antigens entrapped within certain archaeosome compositions give humoral immune responses that are comparable to those obtained with the potent but toxic Freund's adjuvant. Archaeosome compositions can be selected to give a prolonged, sustained immune response, and the generation of a memory response. Tissue distribution studies of archaeosomes administered via various systemic and peroral routes indicate potential for targeting to specific organs. All in vitro and in vivo studies performed to date indicate that archaeosomes are safe and do not invoke any noticeable toxicity in mice. The stability, tissue distribution profiles, and adjuvant activity of archaeosome formulations indicate that they may offer a superior alternative to the use of conventional liposomes, at least for some biotechnology applications.     

The Use of Liposomes as Carriers of Lipophilic Anthracycline Antibiotics

Abstract

Tumor drug resistance and lack of tumor selectivity are the two main limitations of current systemic anticancer therapy. Liposomes have been shown to decrease certain doxorubicin (Dox)-related toxicities. By modifying liposome size and composition, the tumor localization of liposome entrapped drugs can be greatly enhanced. Through extensive structure-activity studies aimed at identifying anthracycline antibiotics which combine an enhanced affinity for lipid membranes and an ability to overcome multidrug resistance (MDR), we have identified Annamycin (Ann), which is ideally suited for entrapment in liposomes of different size and composition and has shown remarkable in vivo antitumor activity in different tumor models that display natural or acquired resistance to Dox. The unprecedented liposome formulation flexibility offered by Ann is expected to facilitate current efforts aimed at developing pharmaceutically acceptable liposomal-Ann formulations with optimal tumor targeting properties.     

Novel camptothecin analogue (gimatecan)-containing liposomes prepared by the ethanol injection method

Small-sized liposomes have several advantages as drug delivery systems, and the ethanol injection method is a suitable technique to obtain the spontaneous formation of liposomes having a small average radius. In this paper, we show that liposomal drug formulations can be prepared in situ, by simply injecting a drug-containing lipid(s) organic solution into an aqueous solution. Several parameters should be optimized in order to obtain a final suitable formulation, and this paper is devoted to such an investigation. Firstly, we study the liposome size distributions determined by dynamic light scattering (DLS), as function of the lipid concentration and composition, as well as the organic and aqueous phases content. This was carried out, firstly, by focusing on POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) then on the novel L-carnitine derivative PUCE (palmitoyl-(R)-carnitine undecyl ester chloride), showing that it is possible to obtain monomodal size distributions of rather small vesicles. In particular, depending on the conditions, it was possible to achieve a population of liposomes with a mean size of 100 nm, when a 50 mM POPC ethanol solution was injected in pure water; in the case of 50 mM PUCE the mean size was around 30 nm, when injected in saline (0.9% NaCl). The novel anticancer drug Gimatecan, a camptothecin derivative, was used as an example of lipophilic drug loading by the injection method. Conditions could be found, under which the resultant liposome size distributions were not affected by the presence of Gimatecan, in the case of POPC as well as in the case of PUCE. To increase the overall camptothecin concentration in the final liposomal dispersion, the novel technique of "multiple injection method" was used, and up to a final 5 times larger amount of liposomal drug could be reached by maintaining approximately the same size distribution. Once prepared, the physical and chemical stability of the liposome formulations was satisfactory within 24, as judged by DLS analysis and HPLC quantitation of lipids and drug. The Gimatecan-containing liposomes formulations were also tested for in vitro and in vivo activity, against the human nonsmall cell lung carcinoma NCI-H460 and a murine Lewis lung carcinoma 3 LL cell lines. In the in vitro tests, we did not observe any improvement or reduction of the Gimatecan pharmacological effect by the liposomal delivery system. More interestingly, in the in vivo Lewis lung carcinoma model, the intravenously administration of liposomal Gimatecan formulation showed a mild but significant increase of Tumor Volume Inhibition with respect to the oral no-liposomal formulation (92% vs. 86 %, respectively; p < 0.05). Finally, our study showed that the liposomal formulation was able to realize a delivery system of a water-insoluble drug, providing a Gimatecan formulation for intravenous administration with a preserved antitumoral activity.     

Biophysical and lipofection studies of DOTAP analogs

In order to investigate the relationship between lipid structure and liposome-mediated gene transfer, we have studied biophysical parameters and transfection properties of monocationic DOTAP analogs, systematically modified in their non-polar hydrocarbon chains. Stability, size and (by means of anisotropy profiles) membrane fluidity of liposomes and lipoplexes were determined, and lipofection efficiency was tested in a luciferase reporter gene assay. DOTAP analogs were used as single components or combined with a helper lipid, either DOPE or cholesterol. Stability of liposomes was a precondition for formation of temporarily stable lipoplexes. Addition of DOPE or cholesterol improved liposome and lipoplex stability. Transfection efficiencies of lipoplexes based on pure DOTAP analogs could be correlated with stability data and membrane fluidity at transfection temperature. Inclusion of DOPE led to rather uniform transfection and anisotropy profiles, corresponding to lipoplex stability. Cholesterol-containing lipoplexes were generally stable, showing high transfection efficiency at low relative fluidity. Our results demonstrate that the efficiency of gene transfer mediated by monocationic lipids is greatly influenced by lipoplex biophysics due to lipid composition. The measurement of fluorescence anisotropy is an appropriate method to characterize membrane fluidity within a defined system of liposomes or lipoplexes and may be helpful to elucidate structure-activity relationships.     

Lipid Dependent Cardio- Haemodynamic Tolerability of Liposomes in Rats

Abstract

Rationale and Objectives:

The use of contrast-carrying liposomes in diagnostic applications (1) or of haemoglobin liposomes in blood replacement therapy (2) requires infusion of large lipid doses. Saturated lipids like HSPC are often used in these formulations to render the liposomes more stable (3). Previous studies have indicated that intravenous injection of such liposome preparations can result in significant haemodynamic changes in rats (14). The purpose of this study was to systematically evaluate cardio- and haemodynamic effects of liposomes prepared from saturated and unsaturated phosphatidylcholine alone or in combination with other lipid components.

Methods;

Liposomes made from SPC, HSPC, DSPC, DSPC/CH, DSPC/DSPG, DSPC/CH/DSPG were infused in anaesthetized rats (total lipid dose: 300 mg lipid/kg BW) and cardio-heamodynamic parameters were measured.

Results:

DSPC-liposomes significantly reduced blood pressure (BP) and total peripheral resistance (TPR) by -53.7 % and -45.7 % of prevalue, respectively. Similar results were obtained for HSPC-liposomes. Marked ECG-changes were recorded for both formulations. SPC-liposomes caused a transient and moderate reduction of BP and TPR (-17.0 % and -22.3 %, respectively). Short-lasting ECG changes were also observed. The addition of cholesterol or DSPG to DSPC liposomes reduced cardiac and haemodynamic side effects in rats.

Conclusion;

The lipid composition of liposomes is of major importance for the incidence of cardiovascular side effects in rats. Liposomes composed of pure saturated phosphatidylcholine cause significant changes which can be diminished by the addition of other lipid components like cholesterol.     

Self-assembly of mixtures of a dendrimer and lipids: effects of hydrophobicity and electrostatics

Self-assemblies of mixtures of a G7 dendrimer and dimyristoylphosphatidylcholine (DMPC) lipids were simulated using the coarse-grained force fields. A single G7 dendrimer, which consists of either a hydrophilic or a hydrophobic interior, was simulated with the randomly distributed zwitterionic DMPC or anionic lipids. For the dendrimer with hydrophilic interior, its mixture with anionic lipids self-assembles to the dendrimer-encasing liposome, but the one with zwitterionic lipids does not. The liposome diameter agrees with experiment, which is smaller than the typical liposome without dendrimer. This indicates that the strong electrostatic interactions between dendrimer terminals and lipid phosphates induce high curvature of the bilayer, leading to such a small liposome. For the dendrimer with hydrophobic interior, lipids penetrate the dendrimer interior because of the hydrophobic interactions between the dendrimer interior and lipid tails, leading to the dendrimer–lipid micelle with increased dendrimer size. These simulation findings agree qualitatively with the experimentally proposed liposome and micelle models, and indicate that these proposed conformations of the dendrimer–lipid complexes are significantly modulated by dendrimer hydrophobicity and lipid charge.     

Stability of pressure-extruded liposomes made from archaeobacterial ether lipids

Ether lipids were obtained from a wide range of archaeobacteria grown at extremes of pH, temperature, and salt concentration. With the exception ofSulfolobus acidocaldarius, unilamellar and/or multilamellar liposomes could be prepared from emulsions of total polar lipid extracts by pressure extrusion through filters of various pore sizes. Dynamic light scattering, and electron microscopy revealed homogeneous liposome populations with sizes varying from 40 to 230 nm, depending on both the lipid source and the pore size of the filters. Leakage rates of entrapped fluorescent or radioactive compounds established that those archaeobacterial liposomes that contained tetraether lipids were the most stable to high temperatures, alkaline pH, and serum proteins. Most ether liposomes were stable to phospholipase A2, phospholipase B and pancreatic lipase. These properties of archaeobacterial liposomes make them attractive for applications in biotechnology.     

A method to determine the incorporation capacity of camptothecin in liposomes

The purpose of this study was to establish a new experimental approach to determine the maximum amount of campothecin (CPT) that can be incorporated in liposomes, and to use this method to compare the CPT-incorporation capacity of various liposome formulations. Small, CPT-saturated liposomes were prepared by dispersing freeze-dried blends of lipids and drug in phosphate buffer, and subsequent probe-sonication. Excess precipitated CPT could be separated from the liposomes by ultra-centrifugation. The small and homogeneous liposome size obtained gave a good and reproducible recovery of liposomes in the supernatant (>80%), whereas the acidic pH (pH 6.0) kept CPT in its hydrophobic lactone form, which is poorly soluble in the buffer. The maximum CPT-incorporation capacity of 12 different liposome formulations was investigated, using the described method, and was found to vary widely. With liposomes made of neutral and anionic phospholipids, the solubili ty of CPT in the buffer was improved by approximately a factor of 10 (from ∼2.7 to 15–50 μg/mL) as compared with buffer. With cationic liposomes containing 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP), a maximum CPT-solubilization of ∼100-fold, the buffer solubility was reached, probably owing to an electrostatic interaction between the cationic lipids and the carboxylate-CPT isomer. Increasing DOTAP fractions within egg-phosphatidylcholine (EPC)/DOTAP liposomes reached a CPT-incorporation plateau at ∼20 mol% DOTAP. The presented approach appears suitable to study the incorporation capacity of any drug component within small vesicles as long as the liposome incorporation is high relative to the intrisic water solubility of the drug.     

Terbinafine hydrochloride loaded liposome film formulation for treatment of onychomycosis: in vitro and in vivo evaluation

Onychomycosis is a fungal infection of nail unit that is caused by dermatophytes. Oral Terbinafine hydrochloride (TBF-HCl) is being used for the treatment of onychomycosis since 24 years. The side effects caused by the systemic application and limitations of topical administration of this drug regarding the diffusion through nail lead to the development of a new formulation based on, TBF-HCl-loaded liposome. The newly obtained film formulations were prepared and characterized via several parameters, such as physical appearance, drug content, thickness, bioadhesive properties and tensile strength. In vitro and ex vivo permeation studies were performed to select an optimum film formulation for antifungal activity to show the efficiency of formulations regarding the treatment of onychomycosis. The in vitro release percentages of drug were found 71.6?±?3.28, 54.4?±?4.26, 56.1?±?7.48 and 46.0?±?2.43 for liposome loaded pullulan films (LI-P, LII-P) and liposome loaded Eudragit films (LI-E, LII-E), respectively. The accumulated drug in the nail plates were found 31.16?±?4.22, 24.81?±?5.35, 8.17?±?1.81 and 8.92?±?3.37 for LI-P, LII-P, LI-E and LII-E, respectively, which within therapeutic range for all film formulations. The accumulated drug in the nail plate was found within therapeutic range for all film formulations. The efficacy of the selected TBF-HCl-loaded liposome film formulation was compared with TBF-HCl-loaded liposome, ethosome, liposome poloxamer gel and ethosome chitosan gel formulations. It was found that TBF-HCl-loaded liposome film formulation had better antifungal activity on fungal nails which make this liposome film formulation promising for ungual therapy of fungal nail infection.     

Lipid analysis of Greek walnut oil (Juglans regia L.)

The walnut oil (Juglans regia L.) total lipids (TL) were extracted by the Bligh-Dyer method and the lipid classes have been isolated by chromatographic techniques and they were analyzed by high performance thin layer chromatography (HPTLC)/FID and GC-MS. The oil was found to be rich in neutral lipids (96.9% of total lipids) and low in polar lipids (3.1% of total lipids). The neutral lipid fraction consisted mainly of triacylglycerides whereas the polar lipids mainly consisted of sphingolipids. GC-MS data showed that the main fatty acid was linoleic acid. Unsaturated fatty acids were found as high as 85%, while the percentage of the saturated fatty acids was found 15%. Two types of liposomes were prepared from the isolated walnut oil phospholipids and characterized as new formulations. These formulations may have future applications for encapsulation and delivery of drugs and cosmetic active ingredients.     

Application of ITC in foods: A powerful tool for understanding the gastrointestinal fate of lipophilic compounds

BackgroundIsothermal titration calorimetry (ITC) is a biophysical technique widely used to study molecular interactions in biological and non-biological systems. It can provide important information about molecular interactions (such as binding constant, number of binding sites, free energy, enthalpy, and entropy) simply by measuring the heat absorbed or released during an interaction between two liquid solutions.Scope of the reviewIn this review, we present an overview of ITC applications in food science, with particular focus on understanding the fate of lipids within the human gastrointestinal tract. In this area, ITC can be used to study micellization of bile salts, inclusion complex formation, the interaction of surface-active molecules with proteins, carbohydrates and lipids, and the interactions of lipid droplets.Major conclusionsITC is an extremely powerful tool for measuring molecular interactions in food systems, and can provide valuable information about many types of interactions involving food components such as proteins, carbohydrates, lipids, surfactants, and minerals. For systems at equilibrium, ITC can provide fundamental thermodynamic parameters that can be used to establish the physiochemical origin of molecular interactions.General significanceIt is expected that ITC will continue to be utilized as a means of providing fundamental information about complex materials such as those found in foods. This knowledge may be used to create functional foods designed to behave in the gastrointestinal tract in a manner that will improve human health and well-being. This article is part of a Special Issue entitled Microcalorimetry in the BioSciences — Principles and Applications, edited by Fadi Bou-Abdallah.